Abstract: Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438– > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. Key points: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68–14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2 Graphical Abstract: [Figure not available: see fulltext.]@en

Sialic acids are a family of nine-carbon negatively charged carbohydrates. In animals, they are abundant on mucosa surfaces as terminal carbohydrates of mucin glycoproteins. Some commensal and pathogenic bacteria are able to release, take up and catabolize sialic acids. Recently, sialic acids have been discovered to be widespread among most microorganisms. Although the catabolism of sialic acids has been intensively investigated in the field of host-microbe interactions, very limited information is available on microbial degradation of sialic acids produced by environmental microorganisms. In this study, the catabolic pathways of sialic acids within a microbial community dominated by 'Candidatus Accumulibacter' were evaluated. Protein alignment tools were used to detect the presence of the different proteins involved in the utilization of sialic acids in the flanking populations detected by 16S rRNA gene amplicon sequencing. The results showed the ability of Clostridium to release sialic acids from the glycan chains by the action of a sialidase. Clostridium and Chryseobacterium can take up free sialic acids and utilize them as nutrient. Interestingly, these results display similarities with the catabolism of sialic acids by the gut microbiota. This study points at the importance of sialic acids in environmental communities in the absence of eukaryotic hosts.@en

Glycosaminoglycans (GAGs) are linear heteropolysaccharides containing a derivative of an amino sugar. The possibility of the presence of GAGs in aerobic granular sludge was studied by combining SDS-PAGE with Alcian Blue staining (at pH 2.5 and 1), FTIR, mammalian Hyaluronic acid and sulfated GAG analysis kits, enzymatic digestion and specific in situ visualization by Heparin Red and lectin staining. GAGs, including Hyaluronic acid-like and sulfated GAGs-like polymers were found in aerobic granular sludge. The sulfated GAGs-like polymers contained Chondroitin sulfate and Heparan sulfate/Heparin based on their sensitivity to the digestion by Chondroitinase ABC and Heparinase I & III. Heparin Red and lectin staining demonstrated that, the sulfated GAGs-like polymers were not only present in the extracellular matrix, but also filled in the space between the cells inside the microcolonies. The GAGs-like polymers in aerobic granules were different from those produced by pathogenic bacteria but resemble those produced by vertebrates. Findings reported here and in previous studies on granular sludge described in literature indicate that GAGs-like polymers might be widespread in granular sludge/biofilm and contribute to the stability of these systems. The extracellular polymeric substances (EPS) in granular sludge/biofilm are far more complicated than they are currently appreciated. Integrated and multidisciplinary analyses are significantly required to study the EPS.@en

Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in “Candidatus Accumulibacter phosphatis”, a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of “Ca. Accumulibacter” to produce different types of NulOs. Proteomic analysis showed the ability of “Ca. Accumulibacter” to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of “Ca. Accumulibacter” in particular, and biofilms in general. Key Points: •“Ca. Accumulibacter” has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.@en

The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.@en

Reducing CO2 emission and energy consumption is crucial for the sustainable management of wastewater treatment plants (WWTPs). In this study, an algal-bacterial aerobic granular sludge (AGS) system was developed for efficient carbon (C) assimilation and nitrogen (N)/phosphorus (P) removal without the need for mechanical aeration. The photosynthetic O2 production by phototrophic organisms maintained the dissolved oxygen (DO) level at 3-4 mg/L in the bulk liquid, and an LED light control system reduced 10–30% of light energy consumption. Results showed that the biomass assimilated 52% of input dissolved total carbon (DTC), and the produced O2 simultaneously facilitated aerobic nitrification and P uptake with the coexisting phototrophs serving as a C fixer and O2 supplier. This resulted in a stably high total N removal of 81 ± 7% and an N assimilation rate of 7.55 mg/(g-MLVSS∙d) with enhanced microbial assimilation and simultaneous nitrification/denitrification. Good P removal of 92–98% was maintained during the test period at a molar ∆P/∆C ratio of 0.36 ± 0.03 and high P release and uptake rates of 10.84 ± 0.41 and 7.18 ± 0.24 mg/(g- MLVSS∙h), respectively. Photosynthetic O2 was more advantageous for N and P removal than mechanical aeration. This proposed system can contribute to a better design and sustainable operation of WWTPs using algal-bacterial AGS.@en

Sulfated glycosaminoglycans (sGAG) are negatively charged extracellular polymeric substances that occur in biofilms from various environments. Yet, it remains unclear whether these polymers are acquired from the external environment or produced by microbes in the biofilm. To resolve this, we analyzed the presence of sGAGs in samples of an acidophilic biofilm collected from Sulfur Cave in Puturosu Mountain (Romania), an environment that is largely inaccessible to contamination. A maximum of 55.16 ± 2.06 μg sGAG-like polymers were recovered per mg of EPS. Enzymatic treatment with chondroitinase ABC resulted in a decrease of the mass of these polymers, suggesting the structure of the recovered sGAG is similar to chondroitin. Subsequent FT-IR analysis of these polymers revealed absorbance bands at 1230 cm−1, 1167 cm−1 and 900 cm−1, indicating a possible presence of polysaccharides and sulfate. Analysis of genomic sequences closely related to those predominant in the acidophilic biofilm, contained genes coding for sulfotransferase (an enzyme needed for the production of sGAG), which supports the hypothesis of microbial synthesis of sGAGs within the biofilm.@en

Recently, we isolated two marine strains, F1T and F21T, which together with Kiritimatiella glycovorans L21-Fru-ABT are the only pure cultures of the class Kiritimatiellae within the phylum Verrucomicrobiota. Here, we present an in-depth genome-guided characterization of both isolates with emphasis on their exopolysaccharide synthesis. The strains only grew fermentatively on simple carbohydrates and sulfated polysaccharides. Strains F1T, F21T and K. glycovorans reduced elemental sulfur, ferric citrate and anthraquinone-2,6-disulfonate during anaerobic growth on sugars. Both strains produced exopolysaccharides during stationary phase, probably with intracellularly stored glycogen as energy and carbon source. Exopolysaccharides included N-sulfated polysaccharides probably containing hexosamines and thus resembling glycosaminoglycans. This implies that the isolates can both degrade and produce sulfated polysaccharides. Both strains encoded an unprecedently high number of glycoside hydrolase genes (422 and 388, respectively), including prevalent alpha-L-fucosidase genes, which may be necessary for degrading complex sulfated polysaccharides such as fucoidan. Strain F21T encoded three putative glycosaminoglycan sulfotransferases and a putative sulfate glycosaminoglycan biosynthesis gene cluster. Based on phylogenetic and chemotaxonomic analyses, we propose the taxa Pontiella desulfatans F1T gen. nov., sp. nov. and Pontiella sulfatireligans F21T sp. nov. as representatives of the Pontiellaceae fam. nov. within the class Kiritimatiellae.@en

In this research, extracellular polymeric substances (EPS), such as EPSflocs and EPSgranules, were successfully extracted from activated and aerobic granular sludge, respectively, and tested as bio-based flame retardant materials. Flax fabric was coated by the biopolymeric substances and its flammability was evaluated based on a vertical burning test defined in US Federal Aviation Regulation. Both EPSflocs and EPSgranules coated flax fabrics achieved the self-extinguishment due to effective char formation. In particular, the result of the EPSgranules coated sample met the aviation requirements for the aircraft interior. Moreover, the presence of carbonated hydroxyapatite was identified in EPSgranules char residue by using FTIR and XRD analysis. It can contribute to the self-extinguishing property of the fabric by enhancing char formation. Thermogravimetric analysis also demonstrated that EPSgranules coated flax was able to produce greater amount of char residue and its decomposition rate was significantly reduced. This research indicates that there is a great potential to use this biopolymer as a resource for developing high performance bio-inspired flame retardant materials and contribute to a circular economy.@en

Sialic acids have been discovered in the extracellular polymeric substances (EPS) of seawater-adapted aerobic granular sludge (AGS). Sialic acids are a group of monosaccharides with a nine-carbon backbone, commonly found in mammalian cells and pathogenic bacteria, and frequently described to protect EPS molecules and cells from attack by proteases or glycosidases. In order to further understand the role of these compounds in AGS, lectin staining, genome analysis of the dominant bacterial species, and shielding tests were done. Fluorescence lectin bar-coding (FLBC) analysis showed an overlap with protein staining, indicating presence of sialoglycoproteins in the EPS matrix. Genome analysis gives a positive indication for putative production of sialic acids by the dominant bacteria Candidatus Accumulibacter. FT-IR analysis shows upon selective removal of sialic acids a decrease in carbohydrates, extension of the protein side chain, and exposure of penultimate sugars. Enzymatic removal of sialic acids results in the removal of galactose residues from the EPS upon subsequent treatment with β-galactosidase, indicating a linkage between galactose and sialic acid at the terminus of glycan chains. This work indicates the importance of sialic acids in the protection of penultimate sugar residues of glycoproteins in EPS, and provides basis for future research in the composition of EPS from biofilms and granular sludge.@en

Elucidating the extracellular polymeric substances (EPS) of anammox granular sludge is important for stable nitrogen removal processes in wastewater treatment. However, due to a lack of standardized methods for extraction and characterization, the composition of anammox granule EPS remains mostly unknown. In this study, alkaline (NaOH) and ionic liquid (IL) extractions were compared in terms of the proteins they extracted from different “Candidatus Brocadia” cultures. We aimed to identify structural proteins and evaluated to which extend these extraction methods bias the outcome of EPS characterization. Extraction was focussed on solubilization of the EPS matrix, and the NaOH and IL extraction recovered on average 20% and 26% of the VSS, respectively. Using two extraction methods targeting different intermolecular interactions increased the possibility of identifying structural extracellular proteins. Of the extracted proteins, ∼40% were common between the extraction methods. The high number of common abundant proteins between the extraction methods, illustrated how extraction biases can be reduced when solubility of the granular sludge is enhanced. Physicochemical analyses of the granules indicated that extracellular structural matrix proteins likely have β-sheet dominated secondary structures. These β-sheet structures were measured in EPS extracted with both methods. The high number of uncharacterized proteins and possible moonlighting proteins confounded identifying structural (i.e. β-sheet dominant) proteins. Nonetheless, new candidates for structural matrix proteins are described. Further current bottlenecks in assigning specific proteins to key extracellular functions in anammox granular sludge are discussed.@en

Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.@en

ANaerobic AMMonium OXidation (anammox) is an established process for efficient nitrogen removal from wastewater, relying on anammox bacteria to form stable biofilms or granules. To understand the formation, structure, and stability of anammox granules, it is important to determine the composition of the extracellular polymeric substances (EPS). The aim of this research was to elucidate the nature of the proteins, which are the major fraction of the EPS and were suspected to be glycosylated. EPS were extracted from full-scale anammox granular sludge, dominated by "Candidatus Brocadia", and subjected to denaturing polyacrylamide gel electrophoresis. By further analysis with mass spectrometry, a high abundant glycoprotein, carrying a heterogeneous O-glycan structure, was identified. The potential glycosylation sequence motif was identical to that proposed for the surface layer protein of "Candidatus Kuenenia stuttgartiensis". The heavily glycosylated protein forms a large fraction of the EPS and was also located by lectin staining. Therefore, we hypothesize an important role of glycoproteins in the structuring of anammox granules, comparable to the importance of glycans in the extracellular matrix of multicellular organisms. Furthermore, different glycoconjugates may have distinct roles in the matrix of granular sludge, which requires more in-depth characterization of different glycoconjugates in future EPS studies.@en